This invention relates to novel amino alcohol dehydrogenases, methods for preparing the enzymes, and uses of the enzymes.
Amino acid dehydrogenases, amine dehydrogenases, aminotransferases have been known to convert a carbonyl group to an amino group. Amino acid dehydrogenase reductivel aminates keto acid to amino acid. Only keto acids and amino acids can be substrates for the enzyme (Experiments of Biochemistry, Vol. 11, ed. by Japan Society of Biochemistry, Amino acid metabolism and biological amine (I) 193-218, J. Org. Chem., 55, 5567, 1990; Fermentation and Industry 40, 301-311, 1982). The inventors examined substrate specificity of commercially available amino acid dehydrogenases, such as L-alanine dehydrogenase and L-glutamic acid dehydrogenase, and found that they do not have any enzymatic activity on other amino acid alcohols. In other words, these NAD (H)-dependent amino acid dehydrogenases only act on very limited amino acids. Amine alcohols include many useful compounds like synthetic intermediates for pharmaceuticals, such as serinol. Any enzyme that can be used for synthesizing these amino alcohols has not been reported.
Amine dehydrogenase uses tryptophane-tryptoquinone (TPQ) or TPQ and heme as prosthetic groups, and uses phenazinementasulfate (PMS), an artificial electron carrier, as an electron acceptor. It is independent of AND(H). This enzyme acts on substrates to produce aldehydes. The substrates include aliphatic 1-amine, such as methylamine, propylamine, n-butylamine, or 1,6-diaminohexane, and some of the enzymes act on arylamine such as 2-phenethylamine or tyramine (Biosci. Biotechnol. Biochem. 62: 469-478, 1998). It does not act, however, on amino alcohols, amino acids, and aliphatic 2-amines at all.
Aminotransferase transfers an amino group of an amino acid donor to keto acid, thereby converting the keto acid into amino acid. xcfx89-Amino acid transaminase or the like, among others, are known to produce an amino compound from ketone, not keto acid (Unexamined Published Japanese Patent Application No. (JP-A) Hei 3-103192, WO97/15682, Appl. Microbiol. Biotechnol. 33, 634-640, 1990, Examined Published Japanese Patent Application No. (JP-B) Hei 4-11194).
No alcohol dehydrogenase which converts keto alcohol into amino alcohol has been reported. The enzymes which converts keto alcohol and keto acid into the corresponding amino alcohol and amino acid, those which converts keto alcohol, ketone, and aldehyde into the corresponding amino alcohol and amine, and those which converts keto alcohol, keto acid, ketone, and aldehyde into the corresponding amino alcohol, amino acid, and amine have not been reported. Furthermore, neither methods for producing such enzymes nor uses of the enzymes have been reported.
An objective of the present invention is to provide enzymes that can reversibly catalyze the redox reactions described below, production methods, and uses of the enzymes. 
As a result of the investigation to achieve the above objective, the present inventors isolated microorganisms producing a novel dehydrogenase that converts keto alcohols into amino alcohols, the one that concerts keto alcohols and keto acids into the corresponding amino alcohols and amino acids, the one that converts keto alcohols, ketones, and aldehydes into the corresponding amino alcohols and amines, and the one that converts keto alcohols, keto acids, ketones, and aldehydes into the corresponding amino alcohols, amino acids, and amines. We purified the enzymes and named them amino alcohol dehydrogenases.
The present inventors also established a method for producing amino alcohol dehydrogenase, a method for producing amino alcohol from keto alcohol using the amino alcohol dehydrogenase, a method for producing amino acid from keto acid, and a method for producing amine from ketone or aldehyde.
Specifically, the present invention relates to an amino alcohol dehydrogenase described below, a method for producing it, and its uses.
(1) An amino alcohol dehydrogenase that reductively converts keto alcohol into amino alcohol, and oxidatively converts amino alcohol into keto alcohol,
(2) The amino alcohol dehydrogenase of (1), which reductively converts keto acid into amino acid and oxidatively converts amino acid into keto acid,
(3) The amino alcohol dehydrogenase of (1) or (2), which reductively converts ketone or aldehyde into amine and oxidatively converts amine into ketone or aldehyde.:,
(4) The amino alcohol dehydrogenase of (1), (2) or (3), which is obtainable from a microorganism selected from the group consisting of the genera Streptomyces, Pseudomononas, Burkholdenia, and Arthrobacter,
(5) The amino alcohol dehydrogenase of (4), wherein the microorganism belonging to the genus Streptomyces is selected from the group consisting of the species Streptomyces virginiae, Streptomyces griseus, Streptomyces avidinii, and Streptomyces pseudovenezulae, 
(6) The amino alcohol dehydrogenase of (4), wherein the microorganism belonging to the genus Pseudomononas is,: the species Pseudomonaonas fluorescens or Pseudomonas marginalis, 
(7) The amino alcohol dehydrogenase of (4), wherein the microorganism belonging to the genus Burkholdenia is the species Burkholdenia cepacia, 
(8) The amino alcohol dehydrogenase of (4), wherein the microorganism belonging to the genus Arthrobacter is the species Arthrobacter aurescens, 
(9) An amino alcohol dehydrogenase having the following physicochemical properties:
(a) NAD(H)-dependent;
(b) a molecular weight of a part of the subunit of about 46,000 Da when determined by SDS-polyacrylamide gel electrophoresis, and of the whole molecule of about 100,000 Da when determined by gel filtration;
(c) substrate specificity, such that it acts on amino alcohols, amines, amino acids in the presence of NAD+ to produce keto alcohols, ketones, aldehydes, and keto acids, and acts on keto alcohols, ketones, aldehydes, and keto acids in the presence of NADH and ammonium ions to produce amino alcohols, amines, and amino acids;
(d) thermostability, such that it is relatively stable at 30xc2x0 C. and inactivated at 40xc2x0 C. or higher when heated at pH 7.0 for 30 min;
(e) optimum temperature of about 30xc2x0 C. in reductive amination at pH 7.0;
(f) optimum pH of 10.0 in oxidative deamination and of 7.0 in reductive amination; and
(g) stability, such that its activity is stable in the presence of glycerol or serinol, or phenylmethylsulfonylfluoride, a protease inhibitor,
(10) A method for producing amino alcohol dehydrogenase, the method comprising culturing a microorganism, which produces the amino alcohol dehydrogenase of any one of (1) to (9), and recovering the enzyme from the culture,
(11) A method for producing amino alcohol, the method comprising reacting keto alcohol with the amino alcohol dehydrogenase of any one of (1) to (9) in a reaction system, and recovering the corresponding amino alcohol from the reaction system,
(12) A method for producing amino acid, the method comprising reacting keto acid with the amino alcohol dehydrogenase of any one of (2) to (9) in a reaction system, and recovering the corresponding amino acid from the reaction system,
(13) A method for producing amine, the method comprising reacting ketone and aldehyde with the amino alcohol dehydrogenase of any one of (3) to (9) in a reaction system, and recovering the corresponding amine from the reaction system,
(14) A microorganism producing amino alcohol dehydrogenase of (1), which has the characteristics of the microorganism selected from the group consisting of Arthrobacter aurescens B151 identified by a deposit number of FERM P-17137, Burkholdenia cepacia B033 identified by a deposit number of FERM P-17138, Pseudomonas fluorescens B101 identified by a deposit number of FERMP-17139, Pseudomonas marginalis B102 identified by a deposit number of FERMP-17140, Streptomyces griseus TPC33081 identified by a deposit number of FERM P-17141, Streptomyces avidinii A044 identified by a deposit number of FERM P-17142, and Streptomyces pseudovenezulae A161 identified by a deposit number of FERM P-17143.
The present invention also provides a method for producing keto alcohol, keto acid, ketone, or aldehyde comprising reacting amino alcohol, amino acid, or amine with the amino alcohol dehydrogenase described above. In this method as well as the above methods (11) to (13), the microorganism of (14) or its treated product can be used in place of the amino alcohol dehydrogenase.